The ability of mammalian cells to respond to changes in available oxygen (O2) levels is essential to normal development and physiology. The hypoxia-inducible factors (HIFs) play a central role in these responses by activating the expression of genes involved in glycolysis, angiogenesis, and hematopoiesis, all of which contribute to hypoxic adaptation. HIFs are heterodimers consisting of alpha (HIF-1alpha or HIF-2alpha) subunits. Activation of HIF complexes depends on hypoxic stabilization of the highly related HIF-1alpha and HIF-2alpha subunits, which are degraded by the 26S proteasome under normoxic conditions. We have previously studied a role for ARNT and ARNT2 in angiogenesis and hematopoiesis. Given that elimination of ARNT blocks all HIF activity, the current proposal will elucidate the unique activities of HIF-1alpha and HIF-2alpha in development and disease and address several important questions: What target genes do HIF-1alpha and HIF-2alpha each regulate? Do HIF-1alpha and HIF-2alpha interact with different transcriptional co-activators or repressors? In what cells do both HIF-1alpha and HIF-2alpha function during development and in what cells do they function independently? What unique roles does HIF-2alpha play in adult physiology and disease? We hypothesize that HIF-2alpha regulates unique target genes that distinguish its activity from HIF-1alpha activity during both embryogenesis and tumor progression. To test this hypothesis, we will (1) investigate the unique and overlapping activities of HIF-2alpha and HIF-1alpha by analyzing their unique and/or common target genes, (2) identify the tissue-/cell- specific regulators of HIF-2alpha, (3) determine the degree of overlapping function between HIF-1alpha and HIF-2alpha in vivo using a genetic "knock-in" approach, and (4) use a conditional knock-out allele to assess specific functions of HIF-2alpha in adult mice. The function of HIF-2alpha has been considerably less well studied than that of HIF-1alpha. The proposed experiments are timely, as recent reports suggest that HIF-2alpha, but not HIF-1alpha, is critical to the progression of specific malignancies such as renal clear cell carcinoma, pheochromocytoma, and astrocytoma. The overall goal of our studies is to elucidate how HIF-1alpha and HIF-2alpha differentially regulate hypoxic gene induction and how this contributes to embryogenesis, angiogenesis, and neoplastic disease.